The Stanford Hepatitis C Cooperative Research Center will support an integrated research program focused on defining the immune correlates of protection against HCV in at-risk subjects that spontaneously clear their repeated infections. Cumulative evidence suggests that protective immunity against HCV includes both virus neutralizing (Vn) antibodies and cellular immunity. The availability of a specimen set of longitudinally collected samples from high-risk individuals followed from pre-infection to exposure followed by natural clearance or progression to chronic infection provides an unprecedented opportunity to characterize naturally-occurring immune protection. This Center will focus on the B cell response. Project 1 will analyze the specificity of the B cell responses employing high throughput isolation of Vn human monoclonal antibodies (HMAbs) by single cell cloning and by yeast display, with an emphasis on analyzing the response to the E1 glycoprotein. Functional and biochemical characterization of individual monoclonal antibodies will provide insights into the breadth of specificities of the Vn and non-Vn antibody responses, whether non-Vn antibodies interfere with Vn antibodies, and whether Vn antibodies elicited in subjects with chronic infections are more restrictive in their breadth of reactivity. Project 2 wil first explore the roles of virus-associated lipoproteins, HCV-receptor interactions, cell- to-cell transmission and non-Vn antiviral activity in modulating antibody-mediated functions; and will assess whether specific Vn or non-Vn HMAbs exhibit an antiviral effect through their Fc part by studying their ability to contribute to an antiviral effect through their interaction with Fc recepors and/or intracellular viral neutralization. Second, the project will determine the in vivo function f Vn and non-Vn HCV HMAbs in a human liver chimeric mouse model for acute HCV infection. The potency and breadth of protection of selected Vn HMAbs, single and in combination, and the ability of non-Vn HMAbs to interfere with this protection will be assessed in this small animal model of acute HCV infection. Collectively, the information gained from these studies will provide the basis for rational vaccine design, and provide much needed insights into the molecular specificities of antibodies that should be elicited by immunization.